Investigating DNA Binding and Conformational Variation in Temperature Sensitive p53 Cancer Mutants Using QM-MM Simulations.

The tp53 gene is found to be mutated in 50% of all the cancers. The p53 protein, a product of tp53 gene, is a multi-domain protein. It consists of a core DNA binding domain (DBD) which is responsible for its binding and transcription of downstream target genes. The mutations in p53 protein are responsible for creating cancerous conditions and are found to be occurring at a high frequency in the DBD region of p53. Some of these mutations are also known to be temperature sensitive (ts) in nature. They are known to exhibit partial or strong binding with DNA in the temperature range (298-306 K). Whereas, at 310 K and above they show complete loss in binding. We have analyzed the changes in binding and conformational behavior at 300 K and 310 K for three of the ts-mutants viz., V143A, R249S and R175H. QM-MM simulations have been performed on the wild type and the above mentioned ts-mutants for 30 ns each. The optimal estimate of free energy of binding for a particular number of interface hydrogen bonds was calculated using the maximum likelihood method as described by Chodera et. al (2007). This parameter has been observed to be able to mimic the binding affinity of the p53 ts-mutants at 300 K and 310 K. Thus the correlation between MM-GBSA free energy of binding and hydrogen bonds formed by the interface residues between p53 and DNA has revealed the temperature dependent nature of these mutants. The role of main chain dihedrals was obtained by performing dihedral principal component analysis (PCA). This analysis, suggests that the conformational variations in the main chain dihedrals (ϕ and ψ) of the p53 ts-mutants may have caused reduction in the overall stability of the protein. The solvent exposure of the side chains of the interface residues were found to hamper the binding of the p53 to the DNA. Solvent Accessible Surface Area (SASA) also proved to be a crucial property in distinguishing the conformers obtained at 300 K and 310 K for the three ts-mutants from the wild type at 300 K

Studying early structural changes in SOS1 mediated KRAS activation mechanism

KRAS activation is known to be modulated by a guanine nucleotide exchange factor (GEF), namely, Son of Sevenless1 (SOS1). SOS1 facilitates the exchange of GDP to GTP thereby leading to activation of KRAS. The binding of GDP/GTP to KRAS at the REM/allosteric site of SOS1 regulates the activation of KRAS at CDC25/catalytic site by facilitating its exchange. Different aspects of the allosteric activation of KRAS through SOS1 are still being explored. To understand the SOS1 mediated activation of KRAS, molecular dynamics simulations for a total of nine SOS1 complexes (KRAS-SOS1-KRAS) were performed. These nine systems comprised different combinations of KRAS-bound nucleotides (GTP/GDP) at REM and CDC25 sites of SOS1. Various conformational and thermodynamic parameters were analyzed for these simulation systems. MMPBSA free energy analysis revealed that binding at CDC25 site of SOS1 was significantly low for GDP-bound KRAS as compared to that of GTP-bound KRAS. It was observed that presence of either GDP/GTP bound KRAS at the REM site of SOS1 affected the activation related changes in the KRAS present at CDC25 site. The conformational changes at the catalytic site of SOS1 resulting from GDP/GTP-bound KRAS at the allosteric changes may hint at KRAS activation through different pathways (slow/fast/rare). The allosteric effect on activation of KRAS at CDC25 site may be due to conformations adopted by switch-I, switch-II, beta2 regions of KRAS at REM site. The effect of structural rearrangements occurring at allosteric KRAS may have led to increased interactions between SOS1 and KRAS at both the sites. The SOS1 residues involved in these important interactions with KRAS at the REM site were R694, S732 and K735. Whereas the ones interacting with KRAS at CDC25 site were S807, W809 and K814. This may suggest the crucial role of these residues in guiding the allosteric activation of KRAS at CDC25 site. The conformational shifts observed in the switch-I, switch-II and alpha3 regions of KRAS at CDC25 site may be attributed to be a part of allosteric activation. The binding affinities, interacting residues and conformational dynamics may provide an insight into development of inhibitors targeting the SOS1 mediated KRAS activation

Remdesivir-Bound and Ligand-Free Simulations Reveal the Probable Mechanism of Inhibiting the RNA Dependent RNA Polymerase of SARS-CoV-2

The efforts towards developing a potential drug against the current global pandemic, COVID-19, has increased in the past few months. Drug development strategies to target the RNA dependent RNA polymerase (RdRP) are being tried worldwide. The gene encoding this protein, is known to be conserved amongst positive strand RNA viruses. This enables an avenue to repurpose the drugs designed against earlier reported inhibitors of RdRP. One such strong inhibitor is remdesivir which has been used against EBOLA infections. The binding of remdesivir to RdRP of SARS-CoV-2 has been studied using the classical molecular dynamics and ensemble docking approach. A comparative study of the simulations of RdRP in the apo and remdesivir-bound form revealed blocking of the template entry site in the presence of remdesivir. The conformation changes leading to this event were captured through principal component analysis. The conformational and thermodynamic parameters supported the experimental information available on the involvement of crucial arginine, serine and aspartate residues belonging to the conserved motifs in RdRP functioning. The catalytic site comprising of SER 759, ASP 760, and ASP 761 (SDD) was observed to form strong contacts with remdesivir. The significantly strong interactions of these residues with remdesivir may infer the latter’s binding similar to the normal nucleotides thereby remaining unidentified by the exonuclease activity of RdRP. The ensemble docking of remdesivir too, comprehended the involvement of similar residues in interaction with the inhibitor. This information on crucial interactions between conserved residues of RdRP with remdesivir through in-silico approaches may be useful in designing inhibitors.</p

Exploring the Conformational Dynamics of RNA Dependent RNA Polymerase of SARS-CoV-2 in the Presence of Various Nucleotide Analogues

RNA dependent RNA polymerase (RdRP) from positive stranded RNA viruses has always been a hot target for designing of new drugs as it is responsible for viral replication. The major class of drugs that are targeted against RdRP are nucleotide analogues. An extensive docking and molecular dynamics study describing the role of natural nucleotides (NTPs) and its analogues in imparting an inhibitory effect on the RdRP has been presented here. RdRP simulations in its apo, NTP-bound and analogue-bound form have been performed for a cumulative time of 1.9 μs. The conformational flexibility of the RdRP molecule was explored using Principal Component Analysis (PCA) and Markov State Modeling (MSM) Analysis. PCA inferred the presence of correlated motions along the conserved motifs of the RdRP. The ligand binding motif F and template binding motif G showed motions that are negatively correlated with one another. LYS 551, ARG 553 and ARG 555 which are a part of the motif F appear to form strong interactions with the ligand molecules. ARG 836, a primer binding residue was observed to strongly bind to the nucleotide analogues. The MSM analysis helped to observe different conformational states explored by the RdRP. The ensemble docking of the ligands on the Markov states suggested the involvement of the above residues in ligand interactions. The Markov states obtained clearly demarcated the open and closed conformations. The closed states were observed to have more favorable docking of the ligands. MSM analysis predicted a probable inhibitory mechanism involving the closing of the template entry site by reduction in the distance between the flanking finger and thumb subdomain. </p

Computational Drug Repurposing Studies on the ACE2-Spike (RBD) Interface of SARS-CoV-2

The novel coronavirus is known to enter the cell by binding to the human transmembrane protein Angiotensin-Converting Enzyme 2 (ACE2). The S(Spike)-glycoprotein of the SARS-CoV-2 forms a complex with the ACE2. Thus, the S-glycoprotein is one of the hot targets, as it forms the first line of contact between the virus and the human cell. Drug repurposing would help in identifying drugs that are safe and have no or fewer side effects. Hence, in addition to the Food and Drug Administration (FDA) approved molecules the compounds from natural sources were also considered. The current study includes docking and simulations of the FDA approved molecules and phytochemicals from Indian medicinal plants, targeting the ACE2-Spike protein complex. Rutin DAB10 and swertiapuniside were obtained as the top-ranked drugs from these two databases, respectively. The molecular dynamics simulations of ligand-free, rutin DAB10-bound, and swertiapuniside-bound ACE2-Spike complex revealed crucial ACE2-Spike interface residues forming strong interactions with the two ligands molecules. This may infer, that they may affect the ACE2 and spike binding. The conformational flexibility in the drug-binding pocket was captured using the RMSD-based clustering of the ligand-free simulations. An ensemble docking was performed wherein the two databases were docked on each of the representatives of ACE2-Spike obtained through clustering. The potential phytochemicals identified belonged to Withania somnifera, Swertia chirayita, Tinospora cordifolia, Andrographis paniculata, Piper longum, and Azadirachta indica. The FDA molecules identified were rutin DAB10, fulvestrant, cefoperazone acid, escin, chlorhexidine diacetate, echinacoside, capreomycin sulfate, and elbasvir.</p

Population distribution based on Relative SASA and Relative Δ G_{<i>protein</i>}, % population less stable than WT at 300 K has been given in red color.

<p>Population distribution based on Relative SASA and Relative Δ G_{<i>protein</i>}, % population less stable than WT at 300 K has been given in red color.</p

Free energy of p53 molecule throughout the 30 ns for WT at 300 K (black), V143A (red), R249S (green) and R175H (blue) at 310 K.

<p>Free energy of p53 molecule throughout the 30 ns for WT at 300 K (black), V143A (red), R249S (green) and R175H (blue) at 310 K.</p

Free energy distribution of p53 molecule along principal component 2 calculated using <i>ψ</i> dihedrals at 300 K and 310 K.

<p>Free energy distribution of p53 molecule along principal component 2 calculated using <i>ψ</i> dihedrals at 300 K and 310 K.</p

Side chain SASA behavior for Cysteine 176 (A), Histidine (B), Cysteine 238 (C) and Cysteine 242 (D) at 300 K and 310 for R175H.

<p>Side chain SASA behavior for Cysteine 176 (A), Histidine (B), Cysteine 238 (C) and Cysteine 242 (D) at 300 K and 310 for R175H.</p

Comparison of ^<i>opt</i>ΔΔ<i>G_bind</i> for the p53 <i>ts</i>-mutants against wild type at both 300 K and 310 K.

<p>Comparison of ^<i>opt</i>ΔΔ<i>G_bind</i> for the p53 <i>ts</i>-mutants against wild type at both 300 K and 310 K.</p